The regulation of cyclic 3',5'-nucleotide phosphodiesterase will be investigated utilizing a Ca2 ion-dependent phosphodiesterase from brain as a model system. This phosphodiesterase is deactivated by the chromatographic removal on ECTEOLA-cellulose of a Ca2 ion-dependent regulator (CDR) identified as a heat stable, acidic Ca2 ion-binding protein. The sensitive phosphodiesterase will be purified to homogeneity by conventional enzyme isolation techniques and characterized physically. The interactions between the CDR, Ca2 ion, and the phosphodiesterase will be investigated by the techniques of circular dichroic spectrophotometry, gel filtration chromatography, and analyses of reaction kinetics. The Ca2 ion sensitivity of the activation will be explored at varying multiplicities of CDR to enzyme and the molar ratio of CDR required for the activation of one mole of enzyme determined. Direct Ca2 ion-binding studies and Ca2 ion analyses will be conducted with purified enzyme from which CDR has been removed to determine whether the enzyme per se has binding sites for Ca2 ion. The relative ability of the phosphodiesterase to hydrolyze cyclic AMP and cyclic GMP will be determined at varying concentrations of cyclic nucleotide substrate, Mg2 ion, and CDR. The Ca2 ion minus dependent phosphodiesterase will be investigated for possible physical and kinetic relationships with other forms of cyclic nucleotide phosphodiesterase. An observed dependence on CDR by a Ca2 ion minus dependent adenylate cyclase from brain will be evaluated for relationships with the CDR- dependent phosphodiesterase. Various cloned lines of astrocytoma and neuroblastoma will be tested for the presence of CDR, the CDR-dependent phosphodiesterase, and the CDR-dependent adenylate cyclase. The influence of various drugs, such as the phenothiazines, will be tested on these enzyme activities in cells grown in tissue culture.